CD38 as a prognostic indicator in B cell chronic lymphocytic leukemia

ABSTRACT

The subject invention discloses a method for determining the prognosis and probable clinical course of a subject diagnosed with B-CLL. Specifically, the invention involves comparing CD38 expression in a biological sample from the subject containing B-CLL cells to a baseline level of CD38 expression, wherein an elevated level of CD38 expression in relation to the baseline level of CD38 expression may indicate poor prognosis or aggressive course of disease in the subject. Also disclosed is a method for determining whether the Ig V genes of the B-CLL cells of a B-CLL patient are mutated, comprising comparing CD38 expression in a biological sample from the subject containing B-CLL cells to a baseline level of CD38 expression, wherein a lower level of CD38 expression in relation to the baseline level indicates IG V gene mutation.

STATEMENT OF GOVERNMENT INTEREST

This invention is supported in part by US PHS grant AI 10811 from theNIH NIAID. As such, the U.S. Government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

B cell chronic lymphocytic leukemia (B-CLL) is the most common leukemiain the Western world (Rai K, Patel D: Chronic Lymphocytic Leukemia, inHoffman R, Benz E, Shattil S, Furie B, Cohen H, Silberstein L (eds):Hematology: Basic Principles and Practice (ed 2nd). New York, ChurchillLivingstone, 1995, p 1308). Around 7,500 individuals develop and 5,000die from this disease each year (Landis S H, et al., CA Cancer J Clin48:6, 1998). Age is an important factor, since the incidence of B-CLLincreases linearly with each decade above the age of 40 (Ries L, et al:SEER cancer statistics review 1973-1991: Tables and graphs., in Ries L,et al (eds). Bethesda, NIH, 1994; Rai K R, Clin Geriatr Med 13:245,1997). In addition, gender is relevant, since men outnumber women by anapproximate 2:1 ratio (Catovsky D, et al., Br J Haematol 72:141, 1989)and may have a worse clinical outcome (Id.; Mandelli F, et al., J ClinOncol 5:398, 1987).

Patients with B-CLL follow heterogeneous clinical courses. Some survivefor prolonged periods without definitive therapy, while others dierapidly despite aggressive treatment (Rai K, Patel D: ChronicLymphocytic Leukemia, in Hoffman R, et al. (eds): Hematology: BasicPrinciples and Practice (ed 2nd). New York, Churchill Livingstone, 1995,p 1308; Zwiebel JA, Cheson BD, Semin Oncol 25:42, 1998). While variousstaging systems, most notably the Rai and Binet staging systems, havebeen developed to address this clinical heterogeneity (Rai K R, et al.,Blood 46, 219, 1975; Binet J L, et al., Cancer 48:198, 1981; and Rai K:A critical analysis of staging in CLL, in Gail R, Rai K (eds): ChronicLymphocytic Leukemia. Recent Progress and Future Directions. New York,Alan R Liss, 1987, p 253), they cannot accurately predict whether anearly or intermediate stage patient will experience an indolent oraggressive course of disease. Specifically, since these systems considergross manifestations of the disease, including the level of blood andmarrow lymphocyte counts, the size and distribution of the lymph nodes,the spleen size, the degree of anemia and the patient's blood plateletcount, they can only identify patients with poor prognostic outcome whenthe disease has progressed to a more advanced state.

At the present time, there is no known treatment for B-CLL which hasbeen shown to definitively increase life expectancy. Consequently, onlypatients classified in the advanced stages of B-CLL have been consideredfor aggressive treatment such as chemotherapy, radiation therapy,surgery, immunotherapy or transplantation. These treatments may exact asevere physical and emotional toll on the patient without necessarilyimproving outcome; in some instances, B-CLL patients may even succumbfrom the rigors of treatment rather than from the effects of B-CLL.Patients classified in the early stages of B-CLL, who may be in betterphysical condition to receive more aggressive or experimental treatment,generally receive no treatment as long as the condition remains stable.This is for two reasons. First, currently available therapies do notextend life span. Second, there are currently no reliable indicators ofwhich early stage patients will do well and which will do poorly.Further, the unpredictable course of the disease can make interpretingthe results of clinical trials difficult, as some early stage patientswill follow an indolent course even without the benefit of treatment.

Such drawbacks have led researchers to develop adjuvant prognosticcriteria to be used in conjunction with the Rai and Binet stagingsystems, including several parameters such as lymphocyte doubling time(Montserrat E, et al., Br J Haematol 62:567, 1986), circulating levelsof β2-microglobulin (Di Giovanni S, et al., Acta Haematol 81:181, 1989;Keating M J, et al., Blood 86:606A, 1995 (Abstract)), circulating levelsof soluble CD23 (Sarfati M, et al., Blood 88:4259, 1996), serumthymidine kinase levels (Kallander C F, et al., Cancer 54:2450, 1984;Hallek M, et al., Blood 93:1732, 1999), bone marrow histology (Rozman C,et al., Blood 64:642, 1984), and cytogenetic abnormalities (Juliusson G,et al., N Engl J Med 323:720, 1990).

An accurate prognostic indicator for B-CLL not related to the symptomsof advanced disease would be desirable in the treatment and casemanagement of B-CLL patients. Specifically, a prognostic indicator thatcould be evaluated at the cellular level at the earliest stages of thedisease (before onset of thrombocytopenia, anemia, spleen and liverenlargement, etc.) would help physicians identify which patients wouldprogress to a more advanced state of the disease and allow the option ofmore aggressive or experimental treatment at a much earlier stage.Additionally, clinical trials of new drugs or experimental therapiescould be directed to patients depending upon their prognostic outlook,thereby allowing for more relevant results in clinical trials. Ideally,the expression of such a prognostic indicator would remain constant overthe course of disease.

B-CLL is characterized by the clonal accumulation of CD5⁺ cells(Caligaris-Cappio, et al., J Exp Med 155:623-8, 1982). Although thesecells originally were considered antigen inexperienced “virgin”lymphocytes, recent data indicate that at least half of these casesrepresent expansions of previously-triggered, post germinal center“memory” B cells (Schroeder and Dighiero, Immunol Today 15:288-294,1994; Fais, et al., J Clin Invest 102:1515-1525, 1998). This conclusionis based on the presence of significant numbers of somatic mutations inthe immunoglobulin (Ig) heavy (H) chain variable region (V) genes. In astudy of 83 (64 IgM⁺ and 19 non-IgM⁺) B-CLL cases, the inventor andcolleagues found significant numbers of V_(H) mutations in approximately50% of the IgM⁺ and 75% of the non-IgM+ (IgG and IgA) cases (Fais, etal, supra, 1998). Taken together with newer studies undertaken by theinventor and colleagues, V_(H) and V_(L) sequencing data suggest thatapproximately 60% of B-CLL cases can be considered to be derived frompost-germinal center (GC) memory B-cells. Thus, the inventorhypothesized that B-CLL cases can be divided into two categories, namelycells clonally derived from post-germinal center memory B-cells(hereinafter referred to as “post-GC B cells”) and pre-germinal center Bcells (hereinafter referred to as “pre-GC B cells”), some of which maybe antigen inexperienced “virgin” lymphocytes or activated B cells thatwere transformed without entering a germinal center, and that thesecategories may be relevant to prognosis.

The expression of specific cell surface markers distinguishes subsets ofnormal human B cells that differ in differentiation and activationstages and in biologic properties (Clark and Lane, Ann Rev Immunol9:97-127, 1991). In particular, analyses of CD38 and IgD expression havebeen especially useful in distinguishing B-cells at various stages ofdifferentiation from naive through memory cells (Pascual, et al., J ExpMed 180:329-339, 1994; Zupo, et al., Blood, 88:1365-1374, 1996).

Accordingly, the inventor sought to determine whether the distinctionsbased upon surface membrane phenotype of B-CLL cells (CD38⁺ or CD38⁻) orIg V gene mutation status might predict different clinical courses andoutcomes for B-CLL patients notwithstanding similar staging of thesepatients using conventional staging methods. Undertaking the experimentsdescribed herein, the inventor has discovered a strong correlationbetween CD38 expression and Ig V gene mutation, and a strong independentcorrelation between each of CD38 expression and IgV gene mutation andpatient prognosis. Since CD38 expression in a subject's B-CLL cells maybe easily and relatively inexpensively determined through variousmethods known and commonly used in the art, CD38 expression inparticular may be a valuable prognostic indicator in B-CLL cases andshould aid in the management of B-CLL patients.

SUMMARY OF THE INVENTION

The present invention discloses a method for determining the prognosisof a subject with chronic lymphocytic leukemia (“B-CLL”), comprisingdetermining whether the subject's B-CLL cells have been clonallyexpanded from post-GC B cells (post-germinal center memory B-cells) orpre-GC B cells (pre-germinal center B cells, some of which may beantigen inexperienced “virgin” lymphocytes or activated B cells thatwere transformed without entering a germinal center), wherein clonalexpansion from post-GC B cells may be indicative of an indolent courseof B-CLL in the subject or favorable prognosis, and clonal expansionfrom pre-GC B cells may be indicative of poor prognosis or an aggressivecourse of disease.

In one method of the present invention, CD38 expression of B-CLL cellsin a biological sample from the subject is compared to a baseline levelof CD38 expression of B-CLL cells, wherein an elevated level of CD38expression in relation to the baseline level of CD38 expression mayindicate poor prognosis or aggressive course of disease in the subject.In one embodiment, the percentage of total B-CLL cells which are CD38⁺in the biological sample is compared to a baseline percentage of CD38⁺B-CLL cells, wherein an elevated percentage of CD38⁺ B-CLL cells inrelation to the baseline percentage of CD38⁺ B-CLL cells is indicativeof poor prognosis. In another embodiment of the invention, the densityof CD38 surface membrane expression on the B-CLL cells in a biologicalsample from the subject is compared to a baseline density of CD38surface membrane expression of B-CLL cells, wherein an elevated densityof CD38 surface membrane expression in relation to the baseline densityof CD38 surface membrane expression may indicate poor prognosis.

Also disclosed is a method for determining whether the Ig V genes of theB-CLL cells of a B-CLL patient are mutated, comprising comparing CD38expression of B-CLL cells (either as a function of relative percentageof CD38⁺ B-CLL cells, or as a function of the relative density of CD38surface membrane expression on the B-CLL cells) in a biological samplefrom the subject to a baseline level of CD38 expression, wherein a lowerlevel of CD38 expression in relation to the baseline level of CD38expression indicates IG V gene mutation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts representative flow cytometric profiles of CD38expression on mutated and unmutated CD5⁺/CD19⁺ B-CLL cases. B-CLL caseswere analyzed by flow cytometry after exposure to anti-CD19-APC,anti-CD5-FITC, and anti-CD38-PE monoclonal antibodies. The eventsillustrated were obtained by gating on cells expressing CD19. Densityplots of CD38 and CD5 expression are shown for eight representativeB-CLL cases. The upper four cases had no mutations in either the V_(H)or V_(L) genes, whereas the lower four cases had mutations in the V_(H)and/or V_(L) genes.

FIG. 2 illustrates the percentages of CD38-expressing B-CLL cells amongpatients (n=37) whose Ig V_(H) and V_(L) genes had been sequenced by theinventor and his colleagues. As established by convention, unmutatedcases (⋄) were defined as cases displaying <2.0% differences from themost similar germline gene; mutated cases (⋄) display ≧2% differences.Note that all the cases (17/17) that have ≧30% CD38⁺ B-CLL cells wereunmutated, whereas only three unmutated cases expressed low numbers(<30%) of CD38⁺ B-CLL cells. These comparisons are statisticallysignificant (p=0.00001; Mann-Whitney test).

FIG. 3 depicts survival based on V gene mutation status and CD38expression. Panel A is a Kaplan-Meier plot comparing survival based onthe absence (“unmutated”: •••••) or presence (“mutated”: -) ofsignificant numbers (≧2%) of V gene mutations in 47 B-CLL cases(unmutated: 24 cases; mutated: 23). The median survival of the unmutatedgroup is 9 years; the median survival of the mutated group was notreached in 19 years; p=0.0001; log-rank test). Panel B is a Kaplan-Meierplot comparing survival based on the detection of ≧30% (•••••) or <30%CD38⁺ B-CLL cells (≧30%: 17 cases; <30%: 19). The median survival of the≧30% CD38⁺ group is 10 years; the median survival of the <30% CD38⁺group was not reached in 19 years (p=0.0001; log-rank test).

FIG. 4 depicts survival based on V gene mutation status and CD38expression among B-CLL patients who stratify to the Rai intermediaterisk category. Panel A is a Kaplan-Meier plot comparing V gene mutationstatus with survival among the cases within the Rai intermediate riskcategory (unmutated: 16 cases; mutated: 9). These cases arefrustratingly difficult for clinicians to treat because they can haveeither an indolent course requiring no or minimal therapy, or a rapiddownhill course despite aggressive treatment. The median survival of themutated group is 9 years; the median survival of the unmutated group is17 years (p=0.0007; log-rank test). Panel B is a Kaplan-Meier plotcomparing numbers of CD38⁺ B-CLL cells with survival among the caseswithin the Rai intermediate risk category (≧30%: 11 cases; <30%: 9cases). The median survival of the 30% CD38⁺ group is 10 years; themedian survival of the <30% CD38⁺ group was not reached in 19 years(p=0.0030; log-rank test). None of the patients in the <30% CD38⁺ groupdied during the follow-up period.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for determining the prognosis orprojected clinical course in a subject with chronic lymphocytic leukemia(“B-CLL”). In particular, the method of the present invention disclosesan immunophenotypic prognostic indicator which predicts whether thecourse of disease in a specific B-CLL patient will be aggressive orindolent, thereby aiding the clinician in managing the patient andevaluating the modality of treatment to be used.

Since at the current time there are no known treatments that willdefinitively increase the life expectancy of persons diagnosed withB-CLL, clinicians must balance the rigors of aggressive or experimentaltreatment with the likelihood that such treatment will result intangible benefit to the patient. In fact, some B-CLL patients succumb tothe combined effects of treatment and B-CLL rather than to the effectsof B-CLL alone. Accordingly, more aggressive treatment, such asradiation therapy, chemotherapy, transplants and immunotherapy, hastraditionally been reserved for those B-CLL patients already in theadvanced stages of B-CLL who stage higher in the conventional Rai andBinet staging systems. However, these patients may be the most illequipped to handle the rigors of such treatment.

Additionally, the heterogenous course of B-CLL complicates theevaluation of clinical trials, as it is difficult to distinguishpatients who are effectively responding to the therapy beingadministered from patients who would have never progressed to a moreadvanced stage of the disease regardless of treatment. Accordingly, animmunophenotypic prognostic indicator which is predictive of a patient'sclinical course, notwithstanding the conventional stage of the disease,will aid clinicians in better evaluating treatment options, as well asgreatly enhancing the value of clinical studies by better distinguishingthe effects of treatment.

The present invention generally discloses a method for determining theprognosis of a subject with chronic lymphocytic leukemia (“B-CLL”),comprising determining whether the subject's B-CLL cells have beenclonally expanded from post-GC B cells (post-germinal center memoryB-cells) or pre-BC B cells (pre-germinal center B cells, some of whichmay be antigen inexperienced “virgin” lymphocytes or activated B cellsthat were transformed without entering a germinal center). Clonalexpansion from post-GC B cells may be indicative of an indolent courseof B-CLL in the subject or favorable prognosis, while clonal expansionfrom pre-GC B cells may be indicative of poor prognosis or an aggressivecourse of disease.

Specifically, in the preferred embodiment of the present invention, CD38expression of B-CLL cells in a biological sample from the subject iscompared to a baseline level of CD38 expression of B-CLL cells, whereinan elevated level of CD38 expression in relation to the baseline levelof CD38 expression may indicate poor prognosis or aggressive course ofdisease in the subject. The method may be performed using any tissuecontaining B-CLL cells, including but not limited to spleen, lymphnodes, bone marrow, lymph, a whole blood sample from the subject or awhole blood sample that has been treated and processed to isolate theperipheral blood mononuclear cells (“PBMC”).

In one embodiment, the percentage of total B-CLL cells in the biologicalsample which are CD38⁺ is compared to a baseline percentage of CD38⁺B-CLL cells, wherein an elevated percentage of CD38⁺ B-CLL cells inrelation to the baseline percentage of CD38⁺ B-CLL cells is indicativeof poor prognosis, and a lower percentage of CD38⁺ B-CLL cells inrelation to the baseline percentage is indicative of a favorableprognosis or indolent course of disease. Alternatively, the density ofCD38 surface membrane expression on the B-CLL cells in a biologicalsample from the subject is compared to a baseline density of CD38surface membrane expression on B-CLL cells, wherein an elevated densityof CD38 surface membrane expression in relation to the baseline densitysurface membrane expression may indicate poor prognosis, and a lowerdensity of CD38 surface membrane expression in relation to the baselinedensity of CD38 surface membrane expression may indicate a favorableprognosis or a more indolent course of disease.

In both embodiments, i.e., comparing the relative percentage of CD38⁺B-CLL cells (as a percentage of the B-CLL population in total, such as apercentage of CD5⁺/CD19⁺ lymphocytes) or comparing the relative densityof CD38 surface membrane expression on B-CLL cells in the biologicalsample, the level of CD38 expression may be determined by any methodcurrently known in the art, including any applicable direct or indirectimmunofluorescence technique. Further, where relative density of CD38surface membrane expression on the B-CLL cells in the biological sampleis being determined, mean channel fluorescence may be used. In apreferred embodiment of the invention, the level of CD38⁺ B-CLLexpression is determined using flow cytometry where the cells have beenlabeled with monoclonal antibodies conjugated with fluorescent dyes orenzymes, although visual immunofluorescence or other methods may also beused. In a specific embodiment, PBMCs are analyzed for surfaceexpression of CD19/CD5/CD38 by triple color immunofluorescence usinganti-CD19-APC, anti-CD5-FITC (CD5 being specific to B-CLL cells and CD19being specific to B lymphocytes, although other combinations ofantigens/labeled antibodies or enzymes may be used that are specific tonarrow the analyzed pool to B-CLL cells) and anti-CD38-PE antibodyconjugates. The preferred antibody conjugate is anti-CD38-PE (SimultestLeucoGATE, from Becton Dickinson Immunocytometry Systems, San Jose,Calif.).

Additionally, it is well within the skill of one of ordinary skill inthe art to devise either direct or indirect immunoassay kits (i.e.,ELISA or other kits) which use similar antigen/labeled antibody orenzyme combinations to detect levels of CD38 expression. The relativepercentage of CD38⁺ B-CLL cells in relation to a percentage baseline ofCD38⁺ B-CLL cells, or the relative density of CD38 surface membraneexpression on B-CLL cells in relation to a baseline density of CD38surface membrane expression, may be determined by comparing theresulting color, fluorescence or equivalent reaction with a controlsample having a predetermined percentage of CD38⁺ B-CLL cells or densityof CD38 (or the relevant epitope of CD38), as appropriate.

While the exact relative percentage of CD38⁺ B-CLL cells or density ofCD38 surface membrane expression that defines poor or favorableprognosis, i.e., the baseline level of CD38 expression of the B-CLLcells, is somewhat arbitrary (as the numerical cut off value may beshifted upward or downward with an attendant loss of accuracy in theprognostic utility of the test), in a preferred embodiment of theinvention using the disclosed antibody or one of equivalent avidity andspecificity, as well as the disclosed anti-CD38-PE fluorochrome, therelative percentage of CD38⁺ B-CLL cells indicating poor prognosis isgreater than 15%, more preferably is greater than 20%, even morepreferably is greater than 25% and most preferably is greater than 30%of the total B-CLL cells in the biological sample. The preferred rangemay be affected by the specific anti-CD38 monoclonal antibody (mAB)used, as different mABs will have different binding affinities (avidity)for CD38 and may bind to different epitopes of CD38, as well as by thespecific fluorochrome used. Further, similar variable parameters existin isolating the B-CLL population in the biological sample. That is, theexact preferred baseline may vary depending upon the specific anti-CD5and anti-CD19 mABs used, and the anti-CD5 and anti-CD19 fluorochromeconjugates used, for the reasons noted above regarding variations inavidity and specificity, as well as the efficiency of the mAB conjugatedfluorochrome. Using the mABs and fluorochromes disclosed in theExperimental Details below will yield the preferred baseline range ofCD38 expression as disclosed herein. However, all other elements beingthe same, mABs with a lower avidity will have a correspondinglyincreased baseline range, so that higher densities of CD38 surfacemembrane expression, or increased relative percentages of CD38⁺ B-CLLcells, will be required to establish a poor prognosis or aggressivecourse of disease.

However, it is well within the skill of one of ordinary skill in the artto determine the appropriate CD38 baseline level, by either using theexperimental method disclosed herein (that is, comparing levels of CD38expression among a characterized group of B-CLL patients with a knownclinical course), or by comparing the relative avidity and specificityof the mABs disclosed herein and the mABs used in any particularinstance, as well as the relative efficiency of the particularfluorochrome used, and thereafter deducing the appropriate baseline.

Also disclosed by the present invention is a method for determiningwhether the Ig V genes of the B-CLL cells of a B-CLL patient aremutated, comprising comparing CD38 expression of B-CLL cells in abiological sample from the subject to a baseline level of CD38expression of B-CLL cells, wherein a lower level of CD38 expression inrelation to the baseline level of CD38 expression indicates IG V genemutation. In one embodiment of the invention, CD38 expression of B-CLLcells in the biological sample is compared to the baseline level of CD38expression by comparing the percentage of total B-CLL cells which areCD38⁺ to a baseline percentage of CD38⁺ B-CLL cells, wherein a lowerpercentage of CD38⁺ B-CLL cells in relation to the baseline percentageis indicative of mutated IgV genes of the B-CLL cells. Preferably, thepercentage of CD38⁺ B-CLL cells indicating Ig V gene mutation is 15% orless, more preferably is 20% or less, even more preferably is 25% orless, and most preferably is 30% or less. In another embodiment of theinvention, CD38 expression of the B-CLL cells in the biological sampleis compared to the baseline level of CD38 expression by comparing thedensity of CD38 surface membrane expression of the B-CLL cells inrelation to a baseline of CD38 surface membrane expression, wherein alower density of CD38 surface membrane expression in relation to thebaseline is indicative of mutated IgV genes of the B-CLL cells.

The present invention is described in the following Experimental DetailsSection which is set forth to aid in the understanding of the invention,and should not be construed to limit in any way the invention as definedin the claims which follow thereafter.

EXPERIMENTAL DETAILS SECTION Methods

(i) Patients

The Institutional Review Boards of North Shore University Hospital,Manhasset, N.Y., and Long Island Jewish Medical Center, New Hyde Park,N.Y., approved these studies. The patients in this study are a subset(n=47) of the well-defined cohort (n=64) of randomly chosen, typicalIgM⁺ B-CLL patients described previously (Fais F, et al., J Clin Invest102:1515, 1998). Patients were selected for the present study based onthe availability of detailed clinical histories (Don Monti Division ofMedical Oncology, North Shore University Hospital, and theHematology/Oncology Division, Long Island Jewish Medical Center) and theavailability of DNA sequences for both the Ig V_(H) (Fais F, et al.,supra, J Clin Invest 102:1515, 1998) and V_(L) (Ghiotto et al., inpreparation) genes in each case. The clinical courses of the patientsthat were analyzed in this study were not significantly different fromthose that could not be studied because of lack of sample or follow-up.There were 34 males and 13 females in this group, with a mean age of63.4 years (range: 38-80). The mean ages of the unmutated (mean: 61.3;range 38-79) and mutated (mean: 65.5; range: 47-80) cases or of the ≧30%CD38⁺ (mean: 63.5; range 38-79) and <30% CD38⁺ (mean: 63.6; range 44-79)cases were similar. Fresh or cryopreserved B-CLL cells were availablefor surface marker analyses on 37 patients (20 unmutated and 17mutated). These samples had been obtained at various points in theclinical follow-up of these patients. There were no differences in thetiming of sample acquisition between the unmutated and mutated groups.

(ii) Cellular Immunophenotypic Analysis

The following antibody conjugates were used: anti-CD23-fluoresceinisothiocyanate (FITC; Immunotech, Inc., Westbrook, Me.), goat anti-humanIgD-FITC (Southern Biotechnology Associates, Birmingham, Ala.), andanti-CD5-FITC, anti-CD5-phycoerythrin (PE), anti-CD38-PE,anti-CD19-allophycocyanin, anti-CD45-FITC and anti-CD14-PE (SimultestLeucoGATE; all from Becton Dickinson Immunocytometry Systems, San Jose,Calif.).

Peripheral blood mononuclear cells (PBMC) were separated fromheparinized venous blood by density gradient centrifugation usingFicoll-Paque (Pharmacia LKB Biotechnology, Piscataway, N.J.), and usedeither immediately or after thawing samples that had been cryopreservedwith a programmable cell freezing machine (CryoMed, Mt. Clemens, Mich.).PBMCs were analyzed for surface expression of CD19/CD5/CD38 andCD19/IgD/CD38 and CD19/CD5/CD23 by triple color immunofluorescence (FaisF, et al., J Clin Invest 98:1659, 1996). For the detection ofCD19/CD5/CD38-expressing cells, mAb labeled with the followingfluorochrome were used: anti-CD19-APC, anti-CD5-FITC, and anti-CD38-PE;for the detection of CD19/IgD/CD38-expressing cells, anti-CD19-APC,anti-IgD-FITC, and anti-CD38-PE mAb were used; for the detection ofCD19/CD5/CD23-expressing cells, anti-CD19-APC, anti-CD23-FITC, andanti-CD5-PE mAb were used. Isotype-matched negative controls were usedin all assays to determine positive from negative results. Flowcytometric analyses were performed on a Becton-Dickinson FACS Caliburflow cytometer equipped with argon and red diode lasers. Measurements offorward and side scatter were combined with CD45 and CD14 determinationsto identify lymphocytes and exclude monocytes. The CellQuest softwaresystem was used to acquire and analyze data.

(iii) Preparation of RNA and cDNA Synthesis

Total RNA was isolated from either fresh or cryopreserved B-CLL cellsusing Ultraspec RNA (Biotecx Laboratories, Houston, Tex.) according tothe manufacturer's instructions. Two μg of RNA were reverse transcribedto cDNA using M-MLV reverse transcriptase (GIBCO BRL, Life Technologies,Grand Island, N.Y.) and 20 pmol of oligo dT primer in a total volume of20 μl (Fais, et al., J. Clin. Invest., 102(8), 1998, 1515-1525). Thesereactions were carried out at 42° C. for 1 hr, heated at 65° C. for 10minutes, and then diluted to a final volume of 100 μl.

To determine the sequence of the B-CLL cells, 3 μl of cDNA wereamplified using a sense V_(H) leader family specific primer inconjunction with an antisense 19mer C_(H) primer. The reaction wascarried out in 50 μl using 20 pm of each primer and cycled with a PerkinElmer Cetus 9600 apparatus (Emeryville, Calif.) as follows: denaturationat 94° C. for 30 sec.; annealing at 55° C. for 30 sec.; and extension at72° C. for 1 min. After 35 cycles, extension was continued at 72° C. foran additional 10 min.

The V_(H) PCR product was either sequenced in both directions by thedideoxy-chain termination method using fluorescent-labeled ddNTP and TAQpolymerase (Applied Biosystems, I System, Foster City, Calif.) and anautomated sequencer (Applied Biosystems, Foster City, Calif.).Sequencing was performed either directly after purification of PCRproducts with Wizard PCR Preps (Promega, Madison, Wis.), or aftercloning into TA vector (Invitrogen, San Diego, Calif.). When the cloningapproach was used, multiple colonies were chosen randomly and sequenced.

The B-CLL sequences obtained were compared with those in the V BASEdirectory using MacVector software, version 6.0 (Eastman Kodak Co., NewHaven, Conn.) to determine the most similar germline VH gene and thedegree of difference from this germline gene. Similar methods were usedto obtain the V_(L) gene sequences and determine the degree ofdifference from the germ line gene. However, V_(L) genes were amplifiedusing a sense V_(L) leader family specific primer in conjunction with anantisense C_(L) primer.

(iv) Statistical Analyses

The percentages of CD5⁺/CD19⁺ B cells that co-expressed CD38 or IgD orCD23 were determined for each patient and statistical differencesbetween the unmutated and mutated groups analyzed using the Mann-Whitneytest. Patients were also classified according to the percentage of B-CLLcells expressing CD38 into ≧30% CD38⁺ and <30% CD38⁺ groups.

To determine the degree of association between the individual patientsbased on the actual percentages of CD38-expressing cells and on the Vgene mutation status or on the percentages of CD23-expressing cells, theSpearman coefficient was calculated. To determine the degree ofassociation between the patients classified into two groups based onpercentages of CD38-expressing cells and on the V gene mutation status,the Kappa coefficient was used. Standard methods for estimatingproportions and associated exact confidence intervals (CI) were used forestimating sensitivity of high numbers of CD38⁺ B-CLL cells (≧30%) as amarker for “unmutated” V genes. In standard epidemiological terminology,the unmutated gene corresponded to the “disease” state and accordinglysensitivity was computed using the number of patients with unmutatedgenes as the denominator. Similarly, specificity of low numbers of CD38⁺B-CLL cells (<30%) was computed using the patients with mutated genes asthe denominator. Accuracy was defined as the percentage of patients whowere classified correctly as unmutated or mutated using the CD38criteria. Positive and negative predictive values were computed usingBayes' Rule.

Comparisons of V gene mutation status and CD38 expression with clinicalcourse were made “blindly”. The investigator who reviewed the clinicalhistories of these patients was unaware of the laboratory data duringthe retrospective chart review. The two-tailed Fisher's Exact test wasused to determine whether chemotherapy requirements, Rai stage atdiagnosis, or gender were significantly associated with V gene mutationstatus or with CD38 percentage. Survival analyses were performed usingthe Kaplan-Meier product-limit method and the log-rank test.

Results

(i) Percentages of CD38⁺ B-CLL Cells Among the Unmutated and MutatedCases

The DNA sequences of the Ig V_(H) (Fais F, et al, supra, J Clin Invest102:1515, 1998) and V_(L) (Ghiotto et al., in preparation) genesexpressed by the leukemic cells of the 47 typical IgM⁺ B-CLL casesincluded in this study were determined previously. Based on the numbersof somatic mutations detected in these genes, the cases were dividedinto two categories: “unmutated” or “mutated”. As per currentconvention, “unmutated” cases were defined as those with <2% differencesfrom the most similar germline gene in both the expressed V_(H) andV_(L) genes; “mutated” cases were defined as those in which the B-CLLcells displayed ≧2% differences in either the expressed V_(H) or V_(L)gene.

To determine whether these genetic differences reflected cellularphenotypic differences, the expression of CD38 and IgD on the B-CLLcells of the 37 patients in whom PBMC were available (20 unmutated and17 mutated) were analyzed. Analyses of CD38 and IgD expression werechosen for these studies because they distinguish B cells at variousstages of differentiation (Clark EA, and Lane P J, Annu Rev Immunol9:97, 1991; Pascual V, et al, J Exp Med 180:329, 1994).

The unmutated and mutated B-CLL cases were similar in CD19⁺ B cellsco-expressing CD5, CD23 and IgD, both in the percentages of positivecells and in mean fluorescence intensity (data not shown). However, thepercentage of CD38⁺ cells was dramatically different between theunmutated and mutated cases. FIG. 1 illustrates eight representativeB-CLL cases analyzed for CD19/CD5/CD38-expressing cells. The V_(H) andV_(L) genes of the four cases listed in the upper panel were notmutated, whereas the V_(H) and/or V_(L) genes in the lower panel weremutated. Note that the unmutated cases have a much higher percentage ofCD38⁺ cells among the CD5⁺/CD19⁺ cells than the mutated cases in thelower panel.

When the percentages of CD38⁺ B-CLL cells in the unmutated and mutatedgroups were compared statistically, very significant differences werefound (means: 63.9% vs. 7.3%, respectively; p=0.00001). The Spearmancorrelation between the individual percentages of CD38⁺ B-CLL cells ineach case vs the actual percentages of V gene mutations was r=−0.75(p<0.001), indicating a relatively strong inverse relationship.Accordingly, while CD38 is clearly an independent marker for predictingclinical outlook, the statistically significant inverse relationshipbetween CD38 expression and V gene mutation indicates that measurementsof CD38 expression can be used to evaluate the level of V gene mutation.A low to moderate direct correlation existed between the percentages ofCD23⁺ B-CLL cells and the percentage of V gene mutation r=0.42; p=0.01).There was no correlation between CD38 expression and CD23 expression(data not shown).

When the results on percentages of CD38 expressing cells were plottedindividually (FIG. 2), the cases could be segregated into two distinctsets, one with ≧30% CD38⁺ cells and the other with <30% CD38⁺ cells. The30% cut off value was chosen empirically based on the observeddistributions on the plot, and it is understood that higher or lower cutoff value may be chosen though such a cut off point may result in loweraccuracy in determining prognosis. Furthermore, an inverse relationshipexisted between CD38 expression and V gene mutation status. The set withthe higher percentages of CD38⁺ B-CLL cells was comprised solely ofunmutated cases, whereas the set with the lower percentages of CD38⁺cells contained all of the mutated cases and three of the unmutatedcases. The Kappa coefficient calculated for association between thesetwo sets of CD38⁺ B-CLL cases vs. the unmutated and mutated groups was−0.84, indicating a strong inverse relationship.

Furthermore, high percentages of CD38⁺ B cells (≧30%) indicated thepresence of <2% mutations with 100% specificity (95% C.I.: 84-100%).Since three patients with unmutated V genes were found to have <30%CD38⁺ B-CLL cells (FIG. 2), the sensitivity of using ≧30% CD38⁺ B-CLLcells as a marker for significant percentages of V_(H) or V_(L) genemutations was 85% (95% C.I.: 62-97%). Based on this specificity andsensitivity and on a prevalence of 60% for ≧2% mutations in either V_(H)or V_(L), the positive predictive value of ≧30% CD38⁺ B cells indicatingthe “unmutated” genotype was 100%. Conversely, the predictive value of<30% CD38⁺ B cells indicating the “mutated” genotype was 82%. These CD38criteria indicate V gene mutation status with 92% accuracy.

The differences in CD38 expression were stable over time and were notinfluenced by chemotherapy. Sixteen patients (7 with CD38 values ≧30%and 9 with <30%) were studied at two or more time points, separated byas much as 6 years. Indeed, the percentages of CD38⁺ B-CLL cellsdetected never differed by more than 10% in any instance. One patientwith 95% circulating CD38⁺ B-CLL cells was studied on five occasionsover a 24 month period and the percentages of CD38⁺ cells detected ineach sample were very similar (<5% difference).

(ii) Clinical Course and Outcome of the Unmutated vs. Mutated B-CLLCases

The treatment histories of the patients with unmutated and mutated Ig Vregion genes were very different (Tbl. 1). Eighteen of the 23 mutatedcases (78.3%) required either no chemotherapy (52.2%) or minimaltreatment (26.1%), while only 20.8% of the unmutated cases required no(16.6%) or minimal therapy (4.2%). These differences were highlysignificant (p=0.0001). Furthermore, 79.2% (19/24) of the unmutatedcases required continuous chemotherapy or chemotherapy utilizing two ormore agents or regimens. Although 18 of these 19 patients (94.7%)received fludarabine, only two achieved a durable clinical response.TABLE 1 Comparison of Treatment Histories Based on Either Ig V GeneMutation Status or the Percentages of CD38⁺ B-CLL Cells TreatmentUnmutated Mutated Patients requiring no or minimal 20.8% (5/24)  78.3%(18/23) treatment* Patients requiring continuous 79.2% (19/24) 21.7%(5/23)  chemotherapy or chemotherapy with 2 or more agents or regimens p= 0.0001† ≧30% CD38+ <30% CD38+ Treatment B-CLL Cells B-CLL CellsPatients requiring no or minimal 23.5% (4/17)  73.7% (14/19) treatment*Patients requiring continuous 76.5% (13/17) 26.3% (5/19)  chemotherapyor chemotherapy with 2 or more agents or regimens p = 0.0067† *Minimaltreatment is defined as less than 6 months of therapy in the years offollow up. †Statistical analyses performed using the two-tailed Fisher'sexact test.

These significant differences in chemotherapy requirements werereflected in significant differences in survival (FIG. 3A). The mediansurvival of the patients in the unmutated group was 9 years, whereasmedian survival for the mutated group was not reached for the durationof follow-up (p=0.0001).

Finally, V gene mutation status was compared with the clinical stage atthe time of diagnosis using the modified Rai system. The patientsstratified to all Rai modified clinical stages at the time of diagnosis(Tbl. 2). Patients who stratify to the Rai intermediate risk group arethe most heterogeneous in treatment requirements and survival andrepresent those in whom outcome is the most difficult to predict (Rai K,et al., supra, in Hematology: Basic Principles and Practice (ed 2^(nd)),p 1308; Zwiebel and Cheson, Semin Oncol, 25:42-59, 1998). Therefore, thesurvival of 25 patients in this group were analyzed (9 mutated cases vs.16 unmutated cases; FIG. 4A). The median survival of the unmutated caseswas 9 years, compared to 17 years for the mutated cases (p=0.0007).TABLE 2 Comparison of Modified Rai Stage at Diagnosis with Ig V GeneMutation Status and the Percentages of CD38⁺ B-CLL Cells Stage UnmutatedMutated Low* 22.7% (5/22)  52.4% (11/21) Intermediate  72.7% (16/22)42.9% (9/21) High  4.6% (1/22)  4.7% (1/21) p = 0.123 ≧30% CD38+ <30%CD38+ B-CLL Cells B-CLL Cells Low† 20.0% (3/15) 50.0% (9/18)Intermediate† 73.3% (11/15) 50.0% (9/18) High  6.7% (1/15)  0.0% (0/18)p = 0.138 *Comparison of V gene mutation status among patients in thelow and intermediate risk categories (p = 0.058; two-tailed Fisher'sExact test). †Comparison of CD38 expression among patients in the lowand intermediate risk categories (p = 0.147; two-tailed Fisher's Exacttest).(iii) Clinical Course and Outcome of B-CLL Cases with ≧30% or <30% CD38⁺Cells.

Since there was a significant correlation between V gene mutation andCD38 expression by the B-CLL cells, chemotherapy requirements andsurvival as a function of the percentages of CD38⁺ leukemic cells werecompared. Significant differences were found for both. Seventy threepercent (14/19) of the <30% CD38⁺ cases required either no or minimalchemotherapy, compared with 23.5% (4/17) of the ≧30% CD38⁺ cases(p=0.0067; Tbl. 1). Conversely, 76.5% of the ≧30% CD38⁺ cases requiredeither continuous chemotherapy or chemotherapy with two or more agentsor regimens.

Median survival for the patients in the ≧30% CD38⁺ group was 10 years(FIG. 3B). In contrast, this value could not be determined for thepatients in the <30% CD38⁺ group since all patients in this group werealive for the duration of follow-up (p=0.0001). Highly significantdifferences in survival also were found among the patients in the Raiintermediate risk group (FIG. 4B). Median survival for the ≧30% CD38⁺patients was reached in 10 years, whereas all patients in the <30% CD38⁺group remained alive throughout the years of follow-up (p=0.003).

(iv) Studies of IgG⁺ and IgA⁺ B-CLL Cases.

The preceding observations were also true for a cohort of non-IgMproducing (IgG or IgA) B-CLL patients (n=16), whose V gene sequenceanalyses were published previously (Fais, et al., supra, J Clin Invest102:1515-25, 1998; Hashimoto, et al., supra, J Exp Med 181:1507-17,1995). The median survival of the unmutated non-IgM cases was only 3years, whereas it was not reached for the mutated cases at 15 years(p=0.004, log-rank test; data not shown). Similar data were obtainedwhen the cases were compared based on CD38 expression, although thesmall numbers of available samples (n=8) precluded accurate statisticalanalysis. When these non-IgM⁺ cases were pooled with the IgM⁺ casesdescribed above (bringing the total number of patients studied to 63),the median survival for the unmutated group (n=29) was 8 years and forthe mutated group (n=34) was not reached for the duration of follow-up(p=0.0001). Similar data were obtained for the CD38 groups: mediansurvival for the ≧30% CD38⁺ (n=19) was 9 years, whereas median survivalfor the <30% CD38⁺ group (n=25) was not reached (p=0.0001).

(v) Gender of the B-CLL Cases Based on Either V Gene Mutation or CD38Expression

The cohort of IgM⁺B-CLL patients in this study consisted of 34 males and13 females (M:F=2.6:1). However, the M:F ratio of the patientsstratified by either V gene mutation status or CD38 expression was verydifferent (Tbl. 3).

In the mutated group, males and females were virtually equallydistributed, whereas in the unmutated group a marked male predominancewas found (M:F ratio=11:1; p=0.003). A similar disparity in genderdistribution was seen when the patients were compared based on thepercentages of CD38⁺ B-CLL cells. The numbers of males and females amongthe <30% CD38⁺ group were almost equal (M:F=1.1:1), whereas malesoutnumbered females in the ≧30% CD38⁺ group (M:F=7.5:1; p=0.031). TABLE3 Gender Differences Based on either Ig V Gene Mutation Status or thePercentages of CD38⁺ B-CLL Cells Unmutated Mutated Male* 91.7% (22/24)52.2% (12/23) Female* 8.3% (2/24) 47.8% (11/23) Male:Female Ratio 11.0:11.1:1 p = 0.003* ≧30% CD38+ <30% CD38+ B-CLL Cells B-CLL Cells Male*88.2% (15/17) 52.6% (10/19) Female* 11.8% (2/17)  47.4% (9/19) Male:Female Ratio  7.5:1 1.1:1 p = 0.031* *Statistically significantdifference in gender distribution between the unmutated and mutatedgroups and between the ≧30% CD38⁺ and <30% CD38⁺ groups (two-tailedFisher's Exact test).

TABLE 4 Characteristics of the Two Groups of B-CLL Patients V Gene % ofCD38+ Chemotherapy M:F Status B-CLL Cells Requirement Survival RatioGroup 1 Unmutated High Extensive Shorter High Group 2 Mutated LowMinimal Longer ˜Equal

Discussion

The preceding data indicate that Ig V gene mutation status and CD38expression are both distinct and important prognostic indicators ofclinical course and outcome in B-CLL. Indeed, those patients in eitherthe unmutated or ≧30% CD38⁺ groups experienced a worse clinical coursethan those patients in the mutated or <30% CD38⁺ groups. This was truefor both chemotherapy requirements (Tbl. 1) and survival (FIG. 3).

Possibly the most clinically relevant correlation was found among thosepatients who presented initially in the Rai intermediate risk category(FIG. 4). These patients are frustratingly difficult for clinicians totreat because they can have either an indolent course requiring no orminimal therapy or a rapid downhill course despite aggressive treatment.Both CD38 expression and V gene mutation status were able to segregatethose Rai intermediate risk patients who followed an indolent coursefrom those whose course was much more aggressive (FIG. 4).

Relevant to observations on Ig V gene mutations and survival is thestudy of Oscier et al (Blood 89:4153-60, 1997), indicating that B-CLLcells with unmutated V_(H) genes frequently contain three copies ofchromosome 12, a cytogenetic marker that is associated with poorclinical outcome. This study was recently extended by Hamblin et al(Blood, 1999 Sep. 15;94(6):1848-54) to a larger cohort of patients.These new data are consistent with the observations herein and showclearly that unmutated V_(H) genes are associated with a more aggressiveform of B-CLL.

When the subject patients were stratified according to V gene mutationstatus or CD38 expression (Tbl. 3), a clear preponderance of males wasnoted in the unmutated and ≧˜30% CD38⁺ (poor outcome) groups (M:F: 11:1and 7.5:1, respectively). These ratios are much higher than thosereported previously (Catovsky, et al., Br J Haematol 72:141-9, 1989).The subject data, however, agree with the studies (Catovsky, et al,supra; Mandelli, et al., supra, J Clin Oncol 5:398-406, 1987) indicatingthat women with B-CLL have a more favorable clinical course than men.Although women comprised only ˜10% of the unmutated and ≧30% CD38⁺ (pooroutcome) groups, they constituted ˜50% of the mutated and <30% CD38⁺(good outcome) groups (Tbl. 3). These data support a role for genderindirectly influencing clinical outcome and possibly B cell maturationand differentiation. The mechanism(s) responsible for these differencesare obscure at this point.

The two sets of B-CLL cases characterized in this study appear torepresent B cells transformed at different stages of B celldifferentiation and/or activation. Thus, those B-CLL cases with mutatedV genes and low numbers of CD38⁺ B-CLL cells are characteristic ofpost-GC, memory B cells (Clark, Annu Rev Immunol 9:97-127, 1991;Pascual, et al., J Exp Med 180:329-39, 1994). Some of these B-CLL cellsmay be derived from the small subset of IgM⁺/IgD⁺ memory cells found inthe blood (Klein, et al, J Exp Med 188:1679-89, 1998) or bone marrow(Paramithiotis and Cooper, Proc Natl Acad Sci USA 94:208-212, 1997) orfrom cells similar to the IgD⁺ memory B cells identified in tonsils(Arpin, et al., J Exp Med 187:1169-78, 1998). Although CD27 is anothermarker that distinguishes pre-GC from post-GC B cells (Klein, et al,supra, J Exp Med 188:1679-89, 1998; Agematsu, et al., J Immunol153:1421-9, 1994; Tangye, et al., J Exp Med 188:1691-703, 1998),differences in CD27 expression among the subject CD5⁺ B-CLL cases werenot found, either in density per cell or in cell number (data notshown). These data are in agreement with those of others (Ranheim, etal, Blood 85:3556-65, 1995; Trentin, et al., Cancer Res 57:4940-7,1997).

In contrast, those B-CLL cases with unmutated V genes and high numbersof CD38⁺ B-CLL cells display surface markers characteristic of B cellsthat have not entered a GC. Since CD38, as detected by mAb conjugatedwith PE (phycoerythrin), is expressed on most blood B cells (Kumagai, JExp Med 181:1101-10, 1995), the ≧30% CD38⁺/unmutated B-CLL cells couldbe derived from either naive B cells or activated B cells that have notentered a GC and have not generated Ig V gene mutations. Based onanalyses of the HCDR3 characteristics of unmutated B-CLL cases (Fais, etal, supra, J Clin Invest 102:1515-25, 1998; Johnson, et al., J Immunol158:235-46, 1997), a favored hypothesis is that some of these unmutatedB-CLL cells have been activated and selected by antigen.

The physiological significance of CD38 expression primarily by theunmutated cases and its potential function in cell survival andproliferation is presently unknown. However, previous studies suggestthat CD38 expression identifies those B-CLL clones that are capable oftransducing signals through their B cell antigen receptors that mayincrease or decrease their chance for survival (Lam, et al., Cell90:1073-83, 1997; Zupo, et al., Eur J Immunol 24:1218-1222, 1994). Inthis regard, Zupo et al have reported that CD38⁺ B-CLL cells can beinduced to undergo apoptosis in vitro after exposure to anti-Igantibodies, whereas CD38⁻ B-CLL cells are resistant to these effects.These data are at variance with the unexpected subject clinicalobservations that those B-CLL cases with higher percentages of CD38⁺ Bcells have a worse clinical outcome. However, it is possible that thequality of the antigen receptor stimulus and the presence of associatedstimuli may lead to diverse endpoints (apoptosis vs. survival).Similarly, triggering through the CD38 molecule can have differenteffects on the survival of B cells depending on the state ofmaturation/activation of the cell. Whereas anti-CD38-mediated signalingresults in the death of immature B cells (Kumagai, supra, J Exp Med181:1101-1-, 1995), mature B cells can be rescued from apoptosis by CD38triggering (Lam, et al., supra, Cell 90:1073-83, 1997; Zupo, et al,supra, Eur J Immunol 24:1218-1222, 1994). Further studies will benecessary to determine how these in vitro data correlate with thesubject clinical observations.

In conclusion, the present studies identify CD38 expression and V genemutation status as novel and independent prognostic indicators thatappear to identify mutually overlapping groups of B-CLL patients (FIGS.1 and 2 and Results). However, since CD38 expression can be determinedmore conveniently and rapidly than Ig V gene mutations, this parametermay be the preferred adjunct to the current staging systems.

Indeed, the results of this simple test should enable physicians topredict with considerable accuracy whether a patient is likely to have afavorable or unfavorable clinical course. Furthermore, since theleukemic cells appear to be fixed in their level of expression of thismarker, determining the percentage of CD38⁺ B-CLL cells may be useful atany point in the clinical course of the individual B-CLL patient.However, the possibility that CD38 expression might change with thealterations in chromosomal structure and gene expression that occur inRichter's transformation cannot be excluded (Koduru, et al., Br JHaematol 85:613-616, 1993).

All publications mentioned herein above are hereby incorporated byreference in their entirety. While the foregoing invention has beendescribed in some detail for purposes of clarity and understanding, itwill be appreciated by one skilled in the art from a reading of thedisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention in the appended claims.

1-14. (canceled)
 15. A method for determining the prognosis of andaggressiveness of disease in a subject with B cell chronic lymphocyticleukemia (“B-CLL”), the method comprising determining whether a bloodsample from the subject comprises B-CLL cells that have Ig V genemutations, wherein the presence of Ig V gene mutations in the B-CLLcells is determined by establishing whether the DNA sequence of eitherthe Ig V_(H) or Ig V_(L) gene of the B-CLL cells of the sample is >2%different from the corresponding non-mutated germline DNA sequence ofthe Ig V_(H) or V_(L) gene, wherein the absence of Ig V_(H) and V_(L)genes in the B-CLL cells that are >2% different from the germline genesin B-CLL cells in the sample indicates an aggressive disease course andpoor prognosis versus an indolent disease course.
 16. The method ofclaim 15, wherein the DNA sequence of the Ig V_(H) and V_(L) genes aredetermined by sequencing cDNA of the Ig V_(H) and V_(L) genes made fromthe B-CLL cells.
 17. A method for evaluating whether a patient with Bcell chronic lymphocytic leukemia (“B-CLL”) should undergo an aggressivecourse of treatment, the method comprising determining whether a bloodsample from the subject comprises B-CLL cells that have Ig V genemutations, wherein the presence of Ig V gene mutations in the B-CLLcells is determined by establishing whether the DNA sequence of eitherthe Ig V_(H or Ig V) _(L) gene of the B-CLL cells of the sample is >2%different from the germline gene that has a DNA sequence that is mostsimilar to the DNA sequence of the Ig V_(H) or V_(L) gene, wherein theabsence of Ig V_(H) and V_(L) genes in the B-CLL cells that are >2%different from the germline genes in B-CLL cells in the sample indicatesthe subject should undergo an aggressive course of treatment.
 18. Themethod of claim 17, wherein the DNA sequence of the Ig V_(H) and V_(L)genes are determined by sequencing cDNA of the Ig V_(H) and V_(L) genesmade from the B-CLL cells.
 19. A method for determining the prognosis ofand aggressiveness of disease in a subject with B cell chroniclymphocytic leukemia (“B-CLL”), the method comprising determining theDNA sequence of the Ig V_(H) and Ig V_(L) gene of the B-CLL cells of thesubject; comparing the DNA sequence of the Ig V_(H) and Ig V_(L) gene ofthe B-CLL cells of the subject with the sequence of the germline IgV_(H) and Ig V_(L) genes that have DNA sequences that are most similarto the DNA sequences of the Ig V_(H) and Ig V_(L) genes from the B-CLLcells; wherein the absence of Ig V_(H) and V_(L) genes in the B-CLLcells that are >2% different from the germline genes in B-CLL cells inthe sample indicates an aggressive disease course and poor prognosisversus an indolent disease course.
 20. The method of claim 19, whereinthe DNA sequence of the Ig V_(H) and V_(L) genes are determined bysequencing cDNA of the Ig V_(H) and V_(L) genes made from the B-CLLcells.